Production of secreted rHtrA proteins in L. lactis. rHtrA1 and rHtrA2 proteins, together with a recombinant form of the staphylococcal nuclease  as a positive control, were produced and secreted in L. lactis. Recombinant strains [MG1363(pLB145) in lanes 1 and 2, MG1363(pVE8126) in lane 3 and MG1363 (pVE8127) in lane 4] were grown to the exponential phase in rich M17 medium supplemented with glucose and buffered with β-glycerophosphate, in flasks. EDTA at 500 μM was added to the cultures to induce (lanes
2–4) or not (lane 1, as a negative control) recombinant protein production. After further growth, culture supernatants were recovered, concentrated, precipitated and finally subjected to SDS-PAGE and Coomassie Brilliant Blue staining. Culture supernatants all show the major lactococcal secreted protein, Usp45  either alone (lane 1) or together with one of the recombinant proteins (lanes 2–4). Usp45 (lanes 1–4), rHtrA1 (lane 3), rHtrA2 (lane 4) and for recombinant staphylococcal nuclease (lane 2), both the secreted form, NucB, and its maturation product (released by lactococcal HtrA protease), NucA [4, 10], are indicated by arrows.